A fluorogenic substrate for isopeptidase T (IPaseT) and other ubiquitin C-terminal hydrolases (UCHs) based on the C-termini of ubiquitin with a kcat/Km value of 18 M⁻¹s⁻¹, which is excited at 340-360 nm and emits at 440-460 nm.
CAT No: R1026
CAS No:167698-68-2
Synonyms/Alias:Z-LRGG-AMC . TFA; UCH Substrate
Z-Leu-Arg-Gly-Gly-AMC is a synthetic peptide substrate widely utilized in biochemical research for its utility in enzymatic activity assays, particularly those involving proteases. Structurally, it consists of a tetrapeptide sequence (Leu-Arg-Gly-Gly) protected at the N-terminus with a benzyloxycarbonyl (Z or Cbz) group and conjugated at the C-terminus to 7-amino-4-methylcoumarin (AMC), a fluorogenic moiety. This configuration allows the substrate to remain non-fluorescent until enzymatic cleavage liberates AMC, resulting in a quantifiable fluorescence signal. The compound's design supports sensitive detection and kinetic analysis of enzyme activity, contributing to its prominence in protease research, inhibitor screening, and mechanistic enzymology.
Enzyme Activity Assays: Z-Leu-Arg-Gly-Gly-AMC serves as a robust fluorogenic substrate for evaluating the catalytic function of serine and cysteine proteases, including those with specificity for arginine or glycine residues. Upon enzymatic cleavage at the peptide bond adjacent to AMC, the released fluorophore emits a strong fluorescent signal, enabling precise, real-time quantification of protease activity. This property makes the substrate highly valuable for kinetic studies, enzyme characterization, and substrate specificity profiling.
High-Throughput Screening: The compound's fluorogenic nature is particularly advantageous in high-throughput screening (HTS) platforms designed to identify and characterize protease inhibitors or modulators. Its compatibility with microplate-based fluorescence detection systems allows for rapid, parallel assessment of numerous samples, facilitating efficient discovery and optimization of bioactive compounds. The substrate's sensitivity and reproducibility support reliable data generation in both academic and industrial screening campaigns.
Mechanistic Enzymology: Researchers employ Z-Leu-Arg-Gly-Gly-AMC to dissect the catalytic mechanisms of proteases by monitoring substrate turnover under various experimental conditions. The real-time fluorescent readout enables detailed analysis of enzyme kinetics, including determination of parameters such as Km, Vmax, and inhibition constants. This mechanistic insight is crucial for understanding enzyme function, substrate recognition, and the effects of mutations or chemical modifications on proteolytic activity.
Protease Profiling: The substrate's specific peptide sequence and AMC conjugation allow for selective profiling of protease subclasses based on their substrate preferences. By incorporating Z-Leu-Arg-Gly-Gly-AMC into multiplexed or comparative assays, researchers can differentiate between protease isoforms, map substrate cleavage sites, and evaluate the impact of environmental factors on protease selectivity. Such profiling supports both fundamental enzymology and the development of targeted biochemical tools.
Analytical Method Development: The predictable and quantifiable fluorescence generated upon substrate cleavage makes Z-Leu-Arg-Gly-Gly-AMC a valuable standard for developing and validating analytical methods in protease research. Its use in calibration curves, assay validation, and inter-laboratory comparisons ensures methodological consistency and accuracy. The substrate's defined chemical properties and performance characteristics contribute to the establishment of robust, reproducible protocols for enzyme quantification and inhibitor assessment.
Through these diverse applications, Z-Leu-Arg-Gly-Gly-AMC supports a wide range of experimental objectives in protease enzymology, assay development, and inhibitor discovery, making it an essential tool for both fundamental research and applied biochemical investigations. Its fluorogenic design and peptide-based specificity enable sensitive, real-time monitoring of enzymatic processes, advancing scientific understanding and technical innovation in the study of proteolytic enzymes.
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